The following documents are believed to represent the current state of the art, and the disclosures of each are incorporated by reference:    1. Tucker, J. A. (2000) The continuing value of electron microscopy in surgical pathology. Ultrastructural Pathology 24:383-9    2. Mathews, R. A. and Donald, A. M. (2002) Conditions for imaging emulsions in the environmental scanning electron microscope. Scanning 24:75-85.    3. Mittler, M. A., Walters, B. C. and Stopa, E. G. (1996) Observer reliability in histological grading of astrocytoma stereotactic biopsies. J. Neurosurg 85:1091-4.    4. Levit-Binnun, N., Lindner A. B., Zik O., Eshhar Z. and Moses, E. (2003) Quantitative detection of protein arrays. Anal. Chem. 75:1436-41.    5. Becker, R. P. and Sogard, M. (1979) Visualization of subsurface structures in cells and tissues by backscattered electron imaging. Scan Electron. Microsc. 1979 (II): 835-70.    6. Sedar, A. W., Silver, M. J. and Ingernan-Wolenski, C. M. (1983) Backscattered electron imaging to visualize arterial endothelial detachment in the scanning electron microscope. Scan. Electron. Microsc. 1983 (II): 969-74.    7. Burns, W. A., Zimmerman, H. J., Hammond, J., Howatson, A. Katz, A. and White, J. (1975) The clinician's view of diagnostic electron microscopy. Hum. Pathol. 6:467-78.    8. Gyorkey, F., Min, K. W., Krisko, I. And Gyorkey, P. (1975) The usefulness of electron microscopy in the diagnosis of human tumors. Hum. Pathol. 6:421-41.    9. Hayat, M. A. (2000) Principles and Techniques of Electron Microscopy—Biological Applications (Fourth edition; Cambridge University Press)    10. Hermann, R., Walther, P. and Müller, M. (1996) Immunogold-labeling in SEM. Histochem. Cell. Biol. 106:31-39.    11. Spargo, B. H. (1975) Practical use of electron microscopy for the diagnosis of glomerular disease. Hum. Pathol. 6:405-20.    12. Gu, X. and Herrera, G. A. (2002) The value of electron microscopy in the diagnosis of IgA nephropathy. Ultrastruct. Pathol 26:203-10    13. Fisher, C, Ramsay, A D, Griffiths, M and McDougall, J. (1985) An assessment of the value of electron microscopy in tumor diagnosis. J. Clin. Pathol. 38:403-8    14. Brocker, W, Pfefferkorn G. (1975) Applications of the cathodoluminescence method in biology and medicine. Scan. Electron. Microsc. 1979;(II):125-32    15. Carlen, B. and Englund, E. (2001) Diagnostic value of electron microscopy in a case of juvenile neuronal ceroid lipofuscinosis. Ultrastruct. Pathol. 25:285-8    16. Hollinshead M, Sanderson J. & Vaux D. J. (1997). Anti-biotin antibodies offer superior organelle-specific labeling of mitochondria over avidin or streptavidin. J Histochem. Cytochem. 45:1053-7    17. Kristiansen, E. and Madsen, C. (1995) Induction of protein droplet (alpha-2 microglobulin) nephropathy in male rats after short-term dosage with 1,8-cineole and 1-limonene. Toxicol. Letters 80:147-52.    18. Goldstein, J. I., Newbury, D. E., Echlin, P., and Joy, D. (1992). Scanning Electron Microscopy and X-ray microanalysis: a text for biologists, matrials scientists, and geologists. Plenum Press, 1992.    19. Schlessinger, J. (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110:669-72.
U.S. patents documents:
U.S. Pat. Nos. 3,218,459; 3,378,684; 4,037,109; 4,071,766; 4,115,689; 4,448,311; 4,587,666; 4,596,928; 4,618,938; 4,705,949; 4,720,622; 4,720,633; 4,880,976; 4,929,041; 4,992,662; 5,103,102; 5,250,808; 5,323,441; 5,326,971; 5,362,964; 5,406,087; 5,412,211; 5,811,803; 5,898,261; 5,945,672; 6,025,592; 6,072,178; 6,114,695; 6,130,434; 6,365,898 and 6,452,177.
Published PCT application WO02/14830-PCT/IL01/00764.
Microscopic examination of biological cells and tissues is a central tool in clinical diagnosis as well as in diverse areas of research in the life sciences. Light microscopy (LM) is performed with thin (several micron) samples, which may include cells, acellular material, or thin layers or sections of tissue, which may be stained with contrast agents such as chemicals or antibodies. Transmission electron microscopy (TEM) usually requires specially prepared ultrathin sections (0.1 micron or less), and reveals a wealth of subcellular information. Each of the aforementioned techniques has limitations: the resolution of light microscopy is limited by diffraction to approximately 0.25 microns and the use of TEM is encumbered by extensive processing of the sample, which may alter its structure significantly. Preparation of samples for standard TEM also requires specific skills and takes at least a few days to achieve. The very thin slices present a very limited, and often arbitrary, portion of the sample, necessitating the imaging of multiple serial sections.
High resolution images can also be achieved by scanning electron microscopy (SEM), in which a focused electron beam scans the sample sequentially, and ensuing signals are used to generate an image. Most often, secondary electrons are detected, yielding information on the surface topography of the sample. Detection of backscattered electrons yields information on material distribution of a region of the sample lying a short distance below the surface, typically up to a few microns. SEM is a reflective mode of imaging, in the sense that electrons do not need to traverse the sample to yield an image. Therefore, samples can be of any thickness, and do not need to be sectioned. However, samples must be placed in a vacuum environment to allow unimpeded motion of the scanning electron beam; therefore, the sample has to be extensively dehydrated and dried. Furthermore, when dried, biological and other organic samples become electrically insulating, leading to artifacts due to charging of the samples by the electron beam. Consequently, samples are usually coated with a conductive layer of carbon or metal.
One approach to observing biological or other wet samples without extensive drying has been the development of environmental SEM and similar techniques. These methods are based on differential pumping and multiple apertures, allowing a localized pressure close to the vapor pressure of water in the close vicinity of the sample, while maintaining a high vacuum through most of the path of the scanning electron beam. In these methods the sample is exposed to a partial vacuum, and maintenance of the hydrated state requires both a low temperature and complicated, manual maintenance of the right pressure. Indeed, the paucity of published reports of biological research using this technique attests to the difficulty in obtaining consistent results of a high standard.
High resolution imaging has wide-spread applications in biology, including imaging of cells, tissues, microbes, and viruses, as well as a-cellular samples such as biological, environmental or industrial fluids, emulsions and suspensions.
A recent review discusses the use of electron microscopy in clinical diagnosis (Tucker J. A., 2000). It is found that in a small but significant proportion of cases (3-8%) proper diagnosis can only be made based on electron microscopy, this is particularly pronounced in oncology and in selected areas such as kidney diseases (Tucker 2000). These numbers are probably an underestimate, since the use of electron microscopy is not primarily limited by lack of utility, but by considerations of cost, the time needed to produce results, and the low throughput. Thus, there is a significant need for an imaging system for biological tissues and cells that achieves electron microscopic resolution with sample preparation procedures comparable with those of light microscopy.